Melanoma is the deadliest form of skin cancer and represents a paradigm for chemo-resistance. We aim to understand resistance mechanisms in melanoma in order to provide the basis for improved targeted therapeutic strategies. The serine/threonine kinase, B-RAF, is somatically mutated in 40-60% of melanomas. Mutant B- RAF hyper-activates MEK-ERK1/2 signaling and is required for melanoma growth and invasion. A phase I clinical trial with the RAF inhibitor, PLX4032 (RG7204/Vemurafenib) yielded promising results in mutant B-RAF melanoma patients. However, 19% of patients were intrinsically resistant to B-RAF inhibitors and most initial responders are now relapsing from acquired drug resistance. An inability to eradicate tumors is also evident using in vivo models in which RAF/MEK inhibitors potently inhibit mutant B-RAF-driven melanoma growth but treatment cessation results in rapid tumor re-growth. The mechanisms underlying resistance to B-RAF inhibitors are unknown and must be elucidated to optimize future clinical trials. Our studies will focus on the stemness factor, FOXD3, which is up-regulated following B-RAF inhibition in mutant B-RAF melanoma cells. In Aim 1 of this application, we will determine the role of FOXD3 in melanoma growth and resistance to B-RAF inhibitors in vivo. In Aim 2, we will identify mechanisms regulating FOXD3 in mutant B-RAF melanomas. In Aim 3, we will determine the role of ERBB3, a direct target of FOXD3, in eliciting the effects of RAF inhibitors. Our experimental approaches are innovative in that we modulate B-RAF activity using clinical grade inhibitors and molecular biology approaches in physiologically relevant in vitro and in vivo models. At the completion of our experiments, we expect to have identified a novel resistance-promoting mechanism and provided evidence for utilizing FOXD3 and/or ERBB3 as a biomarker for B-RAF inhibitor resistant melanoma cells. PUBLIC HEALTH RELEVANCE: Melanoma is the deadliest form of skin cancer and current treatment options are poor. Our application focuses on recent advances in therapeutics designed to target a signaling protein, B-RAF that is expressed in a mutant form in approximately 40-60% of melanomas. An inhibitor of RAF elicits short term clinical benefit in the majority of melanoma patients; however, mechanisms of intrinsic (primary) resistance and acquired (secondary) resistance are apparent. We expect that our results will lead to an understanding of resistance mechanisms in this highly aggressive cancer.